摘要
目的:探索无血清培养的骨髓基质干细胞(BMSCs)向神经细胞诱导分化条件的优化方案,为BMSCs应用于脊髓损伤的临床治疗创造条件。方法:用含2%UtroserG的UltraCULTURE无血清培养体系体外扩增人BMSCs,采用流式细胞仪检测培养细胞的表面标志,再以全反式维甲酸、β-巯基乙醇和神经生长因子为主要成分组成6种诱导液诱导BMSCs向神经细胞分化,镜下观察细胞形态学变化,用抗人β微管蛋白(β-Tubulin)抗体和抗人胶质纤维酸性蛋白(GFAP)抗体进行免疫荧光染色鉴定诱导后的神经细胞,通过流式细胞仪检测诱导后细胞的凋亡率。结果:无血清培养的BMSCs在6种诱导条件下均可不同程度地分化为神经样细胞,镜下可见诱导后细胞表现为神经细胞形态特征,免疫荧光染色显示β-Tubulin和GFAP均有阳性表达,诱导后细胞发生不同程度的凋亡,其中全反式维甲酸和神经生长因子组成的复合诱导液的诱导效率较高,诱导后细胞凋亡率较低。结论:全反式维甲酸与神经生长因子联合应用可在体外高效、稳定地诱导无血清培养的BMSCs分化为神经样细胞,是较佳的诱导条件。
Objective：To study the optimization of induction and differentiation condition of neurocytes from bone marrow stromal cells（BMSCs） cultured in serum-free medium and lay the basis for clinical application of BMSCs on the therapy of spinal cord injury.Metbod：BMSCs were amplified in a serum-free medium（Ultra-CULTURE） supplemented with 2% Utroser G.Cell surface antigens were detected with flow cytometry.Six groups of revulsivum composed of all-trans-retinoic acid,nerve growth factor and beta-mereaptoethol were used to induce BMSCs into neurocytes.Morphological changes during the induction of BMSCs were observed under microscopes.The induced cell were stained immunofluorescently with β-Tubulin and GFAP.The rate of apoptosis of induced cells was evaluated by flow cytometry.Result：BMSCs cultured in serum-free medium could differentiate into neurocytes to some extent after respective induction of 6 groups of revulsivum.The induced cells showed neurocyte-like morphological changes under microscopes.These cells were positively stained with β-Tubulin and GFAP.The induced cells displayed apoptosis to some extent.The group of alltrans-retinoic acid combined with NGF was the optimization of induction and differentiation condition because of its higher inductive efficiency and its lower rate of apoptosis.Conclusion：BMSCs cultured in serum-free medium can effectively and stably differentiate into neurocytes induced by all-trans-retinoic acid combined with NGF in vitro.
出处
《中国脊柱脊髓杂志》
CAS
CSCD
2007年第5期372-375,I0002,共5页
Chinese Journal of Spine and Spinal Cord
基金
自然科学基金资助项目(项目号:30500511)
关键词
骨髓基质干细胞
无血清
诱导分化
神经细胞
凋亡
Bone marrow stromal cells
Serum-free
Differentiation
Neurocyte
Apoptosis
